Isolation and enzyme determination of Candida tropicalis mutants for DCA production

Techniques, named two-step enrichment and double-time replica-plating method (TEDR), are described that allow a mutated population of Candida tropicalis to be enriched efficiently for mutants deficient in the alkane degradation pathway (Alk−) and to be selected easily for mutants increasing in the DCA (dicarboxylic acids) excretion pathway. After C. tropicalis was mutated with ethyl methane sulphonate and ultraviolet, the Alk− mutants were enriched (the first step enrichment, up to eightfold in one round of enrichment) by treatment with nystatin in medium SEL1-1. The mutagen-treated cells were then cultured in medium YPD containing chlorpromazine for further enriching (the second-step enrichment, up to threefold in one round) the mutants with an increasing capacity of α- and ω-oxidation. On the other hand, the Alk− mutants were readily isolated by the SEL1 replica-plating method by using alkane or glucose as the sole carbon source. A total of 43 Alk− mutants were isolated from 2×108 mutagen-treated cells. In the following steps, by using SEL2 replica plating, the screening studies showed that of the 43 Alk− mutants, 11 strains could accumulate DCA greatly from alkane, and strains 1-12 and 1-3, especially, could produce nearly three times as much DCA as the wild-type organism could. The results showed that the strains had more cytochrome P450 activity and a higher converting capacity of alkane.